Project Summary Adoptive T cell therapies that use chimeric antigen receptors (CARs) have shown compelling early success in the treatment of B cell malignancies and have potential for the treatment of other cancers. CARs incorporate important components of the T cell receptor (TCR) signaling pathway within a single transmembrane receptor and contain targeting domains against tumor markers. When expressed in patient T cells, they direct immune functions against cells displaying these markers. A major hurdle currently facing CAR-based therapies is that CARs require high affinity and highly expressed antigens in order to initiate cellular responses in T cells. As a result, CAR therapies have reduced efficacy when cancer cells display low levels of tumor markers or establish immunosuppressive microenvironments, both properties common to solid tumors. Native T cell receptors induce cellular responses to lower affinity and more weakly expressed antigens, and recent published research from the PI's lab implicates the plasma membrane microenvironment as a factor that facilitates immune receptor triggering. Specifically, receptor coupling to ordered membrane domains alters the concentration of kinases and phosphatases close to engaged receptors in the related B cell receptor signaling system, enhancing collective receptor phosphorylation and the sensitivity of the cellular immune response. This proposal aims to build on this past work to improve CAR design by 1) quantifying the local plasma membrane microenvironment surrounding current CARs and TCRs expressed in T cells, and 2) engineering novel CARs with modified transmembrane domains such that CARs more effectively establish local membrane microenvironments that favor their activation. These aims will be accomplished using quantitative super-resolution imaging methods developed in the PI's lab alongside functional studies of TCR and CAR signaling. As current CARs have not been optimized for receptor-membrane interactions, the proposed studies represent a novel approach, enabled by the PI's unique expertise membrane biophysics, protein-lipid interactions, immune signaling, and quantitative super- resolution imaging. The modular design of CARs makes it possible to incorporate improvements resulting from the proposed studies into CARs currently being developed by other laboratories or used in pre-clinical or clinical trials. Overall, these advances represent important steps forward toward realization of the full potential and promise of CAR technology in the fight against cancer.